Cell line Chip DNA
Cells grown at 70% confluency were detached, cross-linked with formaldehyde for 10mins and quenched with 125mM Glycine. Cells were lysed in preparation for in-situ contact generation. Isolated nuclei were permeabilized, restriction digestion was carried out for 2hrs at 37C with MboI (New England Biolabs). Restriction sites filed with dNTP's using Biotin-14-dATP (Jena Biosciences) for 1hr at 37C. The filled ends were then ligated together using a T4 ligase at room temperature for 4 hr with before nuclei were lysed, sonicated (using Covaris) and then H3k27ac (Abcam, ab4729) immunoprecipitation was carried out overnight using antibodies. The morning after, 30ul of beads were added to collect the chromatin-antibody complex then the ChIP DNA was collected, washed and crosslinks reversed overnight using Proteinase K. ChIP DNA was eluted and samples purified using the DNA Clean up kit. The DNA was quantified using a Qubit before biotin ligation junction capture using Streptavidin C-1 beads. Samples were washed and taken forward for Tn5 Tagmentation. Tagmentation and PCR amplification was performed as described. Libraries were size selected to 200–700 bp and sequenced on the NovaSeq 600 using paired-end sequencing. Sequenced reads were processed using the HicPro pipeline (v3.1.0), aligning the data to the hg19 genome version and MboI motif for the analysis.